Note: I have been using subjects suggested by our board of directors for this column for the past two years. I would be happy to attempt covering any subjects that the membership might suggest. Let me hear from you!

What is the best synchrony between IVP embryos and recipients?

There is a great deal of evidence that synchrony between the age of in vivo-derived embryos and the day of the estrous cycle in recipients at the time of transfer is affected very little, if at all, when synchrony is within the three day period of 0, plus one or minus one day (zero, meaning day of estrus and age of embryo are the same). Consequently, the pregnancy rate is not affected when day 7 embryos are transferred into day 6, 7, or 8 recipients. This holds true for both fresh and frozen in vivo-derived embryos (Hasler, 2001).

Is the exposure time of bovine embryos to ethylene glycol (EG) prior to freezing and/or after thawing critical to survival?

John F. Hasler

Efficient and efficacious cryopreservation of bovine embryos is critical to the commercial ET industry because, as shown by the most recent AETA statistics (2011), 72% of embryos were frozen following collection versus only 28% that were transferred fresh into recipients. Following the published report of Voelkel and Hu in 1992 on cryopreservation with EG, the commercial bovine ET industry rather quickly switched from glycerol to EG as the major cryoprotectant in freezing media. The overall percentage of embryos frozen in EG rose rapidly starting in 1992 and reached 97% in 2008, the last year that the AETA collected data on this specific statistic.

During the past 20 years there has been a continuing debate among ET practitioners regarding the question of whether EG is more toxic than glycerol to bovine embryos. This concern has led some practitioners to limit exposure time of embryos to EG for a maximum of 5 min prior to chilling and seeding. I am not aware of any published reports showing that EG is any more toxic to bovine embryos than is glycerol. Voelkel and Hu (1992) reported that 100% of IVF blastocysts exposed for 20 min to 1.5M EG or 1.5M propylene glycol were viable 48 h after being rehydrated in holding medium and cultured in vitro. The authors’ interpretation was that “neither of the cryoprotectants was overtly toxic to bovine embryos”. Also, in the original US patent filed by Voelkel in 1992, an exposure time of 10 to 20 min to EG prior to freezing, at a temperature of 18° to 25°C, was recommended.

Two studies demonstrated that exposure of in vitro-produced (IVP) embryos to EG for up to 40 min (Hasler et al., 1997) or 60 min (Takagi et al., 1993a) prior to freezing did not decrease survival rate of embryos following thawing and return to in vitro culture. Obviously, there is also embryo exposure to EG after thawing and prior to transfer. In an additional study, Takagi et al. (1993b) reported no differences in the survival of IVP embryos exposed to 1.8M EG for 15 min, frozen in a direct transfer protocol and then thawed and held in the straws for <1, 10 and 30 min at 20-25°C before being cultured in vitro. Matoba, et al. (2004) also examined the effect of EG exposure after thawing of IVP embryos and included temperature as another factor. There were no differences in embryo survival after 0, 10, 20 or 30 min exposure at 26.0°C or of 0, 10 or 20 min exposure at 38.5°C. However, at 38.5°C embryo survival decreased when exposure time exceeded 30 min. Lastly, a recent study in Argentina that involved large numbers of in vivo-derived embryos exposed to EG for different periods of time prior to freezing and then in vitro culture after thawing failed to detect an influence of exposure time (Tribulo, et al., 2012). Specifically, blastocyst re-expansion and hatching rates were similar for embryos exposed to EG for 5, 10, 20 and 30 minutes

Enough on in vitro culture, how about pregnancy results of actual transfers into recipients?

Martinez, et al. (2002) reported that there was no significant difference in the pregnancy rates or calving rates of recipients after transfer of in vivo-derived embryos frozen for DT after 5 min versus 20 min exposure to EG. A study in Japan resulted in very similar pregnancy rates after transfer of embryos exposed to EG for periods ranging from 5 to 45 min compared to embryos frozen in glycerol that were thawed and then diluted in a step-wise protocol (Dochi et al. 1998). Contemporary comparisons of exposure time to EG within one laboratory or ET program provide the most powerful evidence. However, comparisons between ET programs also provide some indication that EG is not toxic when exposure time is more than 5 min. In the data set that I collected in 2012 from 5 large commercial ET units in Canada and the US, there was no evidence that exposure time to EG ranging from 4 minutes to 40 minutes had an influence on conception rates (Hasler, J.F., 2012). One last data set was reported from Canada early in the commercial usage of EG for embryo freezing. McIntosh and Hazeleger (1994) reported a pregnancy rate of 59% for a large number of embryos that were exposed to EG for 10 to 20 min prior to freezing in EG.

The data that I have been able to uncover strongly support the principle that toxicity is not an issue when embryos are exposed to EG for periods up to 30 or more minutes. However, there is some evidence that somewhat shorter maximal exposures of 20 min or so should be used at elevated temperatures.

I do not recommend that anyone currently using minimal exposure times of 5 min or so lengthen the timing of their protocol. If a protocol is working well and does not involve serious inconvenience, there is no reason to change, even if science supports the efficacy of different protocols.

Introduction – After our annual conference in Winnipeg, our AETA Board of Directors asked me if I would be willing to start a column in A Closer Look that deals with practical ET problems and provides published documentation for the subjects and issues in question. I agreed to do this and the following is my first ‘column’. All of us hold personal opinions on numerous technical aspects of our ET profession. In some cases these opinions are based on well-established principles. However, in some cases we all base opinions primarily on ‘clinical impressions’. I believe that such impressions can sometimes be entirely accurate, but I also know, from personal experience, that unless one accumulates an adequate data base, clinical impressions can sometimes turn out to be wrong. Consequently, this column will present information that has some published documentation and is based on more than just my personal opinion or, in fact, undocumented opinions period. Our board members have submitted a number of suggested topics and have given me the freedom to pick among them. I will attempt to adequately document any subject that is covered in this column. Also, please feel free to suggest future subjects that you feel are of interest to most of our membership.

For what time span can bovine embryos be maintained between recovery and freezing without compromising viability?

For a 10 year period from the mid-1980s to the mid-1990s, Em Tran, Inc. shipped many thousands of frozen embryos from Holstein donors in the US to Holland Genetics, The Netherlands. During one phase of this program, we kept very precise track of the time, down to the minute, between flushing and freezing, out to a maximum of 3 hr or 180 min. Of a total of 2,102 embryos frozen in glycerol during this period, the pregnancy rate for embryos frozen between 0-30 min after flushing was 54.1% and did not vary significantly among 30 minute blocks of time out to the 150-180 min group, which had a pregnancy rate of 55.7%. These embryos were held at ambient lab temperature in Dulbecco’s modified PBS, which we purchased in the powder form and reconstituted in MilliQ water in our lab, adding antibiotics and 10% FCS. The recipients were primarily Holstein cows.

Another data set generated from 3,570 embryos frozen in the Netherlands at Holland Genetics and transferred within the country showed a slightly different outcome (Otter, 1994). The pregnancy rates were 71%, 66% and 57% for embryos held for 0-2, 2-4 or 4-6 hr respectively. These differences were statistically significant.

I recently uncovered a publication on this subject that I had long forgotten about. At the 6th AETA convention in Orlando FL, in 1987, our former AETA president Stan Coley presented some very interesting frozen embryo data from his ET practice in GA. Stan presented data indicating a very high pregnancy rate (75%) for 300 embryos held in in modified PBS + 0.4% BSA and frozen in glycerol within 4 hr post collection. The pregnancy rate was slightly lower for 76 embryos held at ambient temperatures for 4-8 hr before freezing and lower yet (39%) for a small group of 31 embryos held for over 8 hr. Maintaining a total of 91 embryos at 4°C for over 8 hr did not substantially improve the pregnancy rate (46%) compared to the 8 hr group held at ambient temperature.

Pregnancy rates of frozen-thawed embryos are the best index of embryo viability. However, in vitro culture after thawing also can provide meaningful information. In a study conducted at Virginia Polytech (Jousan, 2003), embryos were recovered from donors, maintained in holding medium (OCM; ECHM-500, Immuno-Chemical Products, Ltd.), at either 5 or 22°C for 2, 6 or 12 hr and then frozen in EG. After thawing the embryos were cultured in a commercial IVC medium and examined after 72 hr of culture. There was no difference in the development rates of embryos held for 2 versus 6 hr prior to freezing (86 vs. 79% respectively), but embryos held for 12 hr had a significantly lower survival rate (54%). Holding embryos at 5 versus 22°C prior to freezing did not improve the survival rates for any of the holding periods.

Unfortunately, none of these studies compared different holding media along with different holding times. Certainly, holding medium quality would enter into this issue as perhaps would temperature if it was highly elevated.

If I was still directly involved with freezing embryos commercially, I would feel most comfortable sticking to a 3-4 hr maximum holding time (in a commercial holding medium) whenever possible. However, a few more hours in holding medium might lower subsequent pregnancy rates but probably would not lead to a ‘disaster’.

Greetings fellow AETA members: As the summer heat settles in, we wish everyone a safe and relaxing season. The year tends to pass quickly, and we continue to gear up for our annual convention in beautiful Colorado Springs. We thank Dr. Matt Dorshorst and his convention planning team for their outstanding efforts on this convention.[…]

Dear AETA members: The purpose of AETA is to unite those organizations and individuals in the United States engaged in the embryo transfer industry into an affiliated federation operating under self- imposed standards of performance and conduct. As AETA delivers on education and standards of quality through its certification program, we become the “voice” for[…]

The 2019 AETA-CETA/ACTE Joint Annual Convention will be held from October 24–26, 2019, in Colorado Springs, Colorado. The conference will take place at the Cheyenne Mountain Resort. All of the AETA scientific, social and exhibitor information can be found on the AETA Annual Convention page as it becomes available. Check back often! Colorado Springs is[…]

The AETA/AASRP 2019 Small Ruminant Embryo Transfer Seminar was held June 19 to 22, 2019, at The Ohio State University Large Animal Services satellite veterinary teaching hospital in Marysville, Ohio. The meeting was organized by Dr. Eric Gordon. The course started on Wednesday, June 19, with a review of small ruminant reproductive physiology by Dr.[…]

The AABP office has received questions from members regarding the legality of using compounded estradiol products in cattle for embryo transfer protocols. AABP has also been in discussion with the FDA about the use of compounded estradiol products in food animals. Compounding from approved drugs in animals is only permitted under the narrowly defined conditions[…]

Read full paper here C. Wrenzycki http://dx.doi.org/10.21451/1984-3143-AR869 Anim Reprod, vol.13, n3, p.279-282, 2016 Abstract Over the past decades in vitro production (IVP) of bovine embryos has been significantly improved. Nevertheless, embryos generated in vitro still differ from their in vivo produced counterparts. Embryos must adjust to multiple microenvironments at preimplantation stages. Consequently, maintaining or mimicking the in[…]

By Dr. Matt Iager, 2019 AETA President Greetings to you from AETA. As spring business gears up, don’t forget to take time to interact with friends and family and thank the farmers and ranchers who provide the avenue for our services. AETA has been busy focusing and planning for a fun and successful 2019! In[…]

The 2019 AETA-CETA/ACTE will be held from October 24–26, 2019, in Colorado Springs, Colorado. The conference will be held at the Cheyenne Mountain Resort. All of the AETA scientific, social, and exhibitor information can be found on the AETA Annual Convention page as it becomes available. Check back often! Colorado Springs is a wonderful city[…]

Submitted by Dr. Kevin Lindell Recently, Dr. Rachael Gately, Tufts Veterinary Field Service, had the opportunity (through a collaborative research project) to explore the realm of small ruminant ovum pick-up (OPU)/in vitro fertilization (IVF). We thought it would be interesting to the membership to relay our initial experiences and challenges. To date, donors have been[…]